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Biomedical subjects

R Tjian

Publications and source records attributed to R Tjian.

At least 199 records · Page 11Linked to original sources
PubMed1981

Does simian virus 40 T antigen unwind DNA?

Recently developed techniques allow rapid and accurate determination of protein-induced DNA unwinding. We have used such techniques to determine whether SV40 T antigen unwinds DNA. Assays were performed with purified wild type T antigen, D2 protein and the SV80 T protein over a wide range of protein concentrations. Additionally, several DNA templates were used, including SV40 DNA, pBR322 DNA, and two plasmid DNAs containing one and three copies of the SV40 origin of replication. None of the purified proteins showed unwinding activity either in the presence or absence of ATP and Mg2+. As a positive control, we found that unwinding of pBR322 DNA by Escherichia coli RNA polymerase occurred under identical assay conditions. Protein-DNA interactions that involve unwinding of the helix are sensitive to the superhelical density of the DNA. Using a filter binding assay, we found that the efficiency of the binding of T antigen to the viral origin of replication does not depend upon the topological state of the DNA. This result further supports our conclusion that SV40 T antigen is not an unwinding protein.

Antigen-Antibody Complex↗
PubMed1981

SV40 gene expression is modulated by the cooperative binding of T antigen to DNA.

We analyzed the DNA binding properties of wild-type simian virus 40 large T antigen and found that the protein binds cooperatively to three tandem sites at a regulatory region of SV40 DNA. One consequence of this T antigen:DNA interaction is the specific repression of SV40 early RNA synthesis in vitro. We mapped a region of 85 base pairs that is necessary and sufficient to initiate early SV40 transcription in vitro. This promoter region lies directly adjacent to the third T antigen binding site but does not include that "TATA" sequence. To determine how T antigen interacts with its binding sites to repress RNA synthesis, we analyzed transcription directed by a variety of wild-type, mutant and hybrid template DNAs. Our findings suggest that the cooperative binding of T antigen to its sites is directly responsible for inhibiting the initiation, rather than blocking elongation, of early RNA synthesis. A model is presented to explain the role of T antigen binding in the regulation of viral transcription and DNA replication during SV40 lytic infection.

Antigens, Neoplasm↗
PubMed1981

Expression of SV40 T antigen under control of adenovirus promoters.

We have obtained novel adenovirus-SV40 recombinant viruses that express wild-type SV40 large T and small t antigens under the control of different adenovirus promoters. Hybrids were constructed in vitro with SV40 DNA that contains the entire early coding region but lacks the transcriptional promoter. Recombinants were isolated by a strong biological selection for viruses that express SV40 T antigen. Analysis of several recombinant genomes indicates that they contain the SV40 A gene inserted in a variety of positions and orientations in the adenoviral genome. Moreover, the set of hybrid transcripts reveals an unexpected variety of splicing patterns. Some hybrid mRNAs transcribed from the adenovirus late promoter appear to contain the adenovirus tripartite leader sequence. Other hybrid mRNAs were transcribed from adenovirus early promoters. All recombinant mRNAs contain intact SV40 early sequences that have normal splice patterns and produce wild-type T antigens. Biochemical characterization of SV40 T antigens overproduced by the hybrid viruses indicates that they are structurally indistinguishable from wild-type SV40 large T antigen and are functionally equivalent to the D2 protein.

Adenoviruses, Human↗
PubMed1981

Properties of simian virus 40 small t antigen overproduced in bacteria.

We constructed a series of bacterial plasmids which contained the Escherichia coli lac promoter fused to a simian virus 40 restriction fragment coding for small t antigen. These plasmids expressed different levels of intact viral protein depending on the length of the constructed ribosome binding site. Small t antigen synthesized by the most efficient producer, HP1, constituted 0.5 to 1% of the total cellular protein. On the basis of extensive characterization by immunoprecipitation, gel electrophoresis, isoelectric focusing, tryptic fingerprint analysis, and chromatographic properties, this plasmid-encoded protein was virtually identical to authentic simian virus 40 small t antigen. Partial purification of the HP1-encoded and authentic small t antigens revealed the presence of both monomeric and multimeric forms.

Antigens, Neoplasm↗
PubMed1980

Regulation of simian virus 40 early transcription in vitro by a purified tumor antigen.

Cloned DNA templates were used to direct the transcription of early and late simian virus 40 (SV40) genes by a cell-free RNA-synthesizing system. Transcription by RNA polymerase II was sensitive to low levels of alpha-amanitin and completely dependent on exogenously added DNA template. RNA products of discrete lengths were efficiently synthesized when transcription was directed by DNA restriction fragments containing promoter sequences for either early or late genes of SV40. Addition of the D2 tumor antigen to the template DNA inhibited transcription originating from the SV40 early promoter. In contrast, the D2 protein had little or no effect on the transcription from SV40 or adenovirus 2 (Ad2) late promoter sequences. When a mixture of cloned DNA containing SV40 early promoter and Ad2 late promoter was used to direct RNA synthesis, the D2 protein specifically inhibited the synthesis of SV40 early genes but not that of Ad2 late sequences. The D2 DNA binding protein also had no effect on the transcription directed by SV40 mutant templates that contain an intact early promoter sequence but lack specific tumor-antigen binding sites. We have confirmed that, under the conditions of the transcription assay, the D2 protein binds and interacts specifically with its recognition sites on wild-type template DNAs but fails to bind to mutant or Ad2 DNA templates that lack sequences containing SV40 tumor-antigen binding sites. These findings provide evidence that a direct interaction between tumor antigen and its specific binding sites on DNA is the mechanism by which the SV40 A gene autoregulates its transcription.

Antigens, Neoplasm↗
PubMed1980

Construction and analysis of simian virus 40 origins defective in tumor antigen binding and DNA replication.

We have inserted a 311-base pair DNA fragment containing the simian virus 40 (SV40) origin of DNA replication, the early promoter, and the tumor (T) antigen binding sites into a bacterial plasmid and cloned it. This recombinant plasmid, pSV01, binds to a purified T antigen in vitro and replicates in monkey cells when supplied with large T antigen. A series of deletion mutations was generated in the origin sequences of pSV01 DNA by mutagenesis in vitro. The replication of these mutant DNAs in monkey cells was compared with their ability to bind to purified D2 protein. Mutant DNAs deficient in binding to D2 protein also exhibit reduced levels of replication in monkey cells. These findings provide biochemical evidence that the initiation of SV40 DNA synthesis may involve a direct interaction of T antigen with sequences at the origin of replication.

Animals↗
PubMed1980

Simian virus 40 small-t protein is required for loss of actin cable networks in rat cells.

The ability of the two early simian virus 40 (SV40) coded proteins, the large and small T-antigens, to abortively induce the disappearance of cytoplasmic actin-containing networks in cultured cells has been studied in rat embryo fibroblasts after microinjection of intact SV40 DNA, DNA fragments from the early region of SV40, and a purified SV40 large T-antigen related protein (the D2 hybrid protein) isolated from cells infected with the adenovirus-SV40 hybrid virus Ad2+D2. Injection of either the 107,000-dalton D2 hybrid protein or SV40 DNA from the deletion mutant dl 884 SV40, which lacks part of the region (0.54 to 0.59) encoding small t-antigen, failed to cause any detectable change in the structure of actin cables in recipient cells over a period of 72 h. By contrast, injection of wild-type SV40 DNA or a DNA fragment containing the entire region coding for a small-t antigen leads to the disruption of actin cable networks within 24 h of injection. It appears likely that the SV40 small-t protein is necessary for the abortive loss of actin cables in injected cells. Epidermal growth factor also causes loss of actin cables in rat embryo fibroblasts or Rat 1 cells (an established rat embryo line), but only after exposure of the cells to epidermal growth factor in the culture medium and not after injection of epidermal growth factor into the cells.

Animals↗
PubMed1979

Enzymatic activities associated with a purified simian virus 40 T antigen-related protein.

A protein antigenically related to the simian virus (SV 40) A gene product has been purified to near homogeneity from cells infected with the adenovirus-SV 40 hybrid virus Ad2(+)D2 and shown to contain ATPase (ATP phosphohydrolase, EC 3.6.1.3) and protein kinase (ATP:phosphotransferase, EC 2.7.1.37) activity. Both enzymatic activities copurify with the protein through six stages including one gel filtration column, two ion exchange columns, and a heparin affinity column. Analogous fractions from extracts of cells uninfected or infected with adenovirus 2 alone do not contain these enzymatic activities. The D2 hybrid protein resolves into two forms (I and II) during ion exchange chromatography. Form I, the major species (85%) of the D2 hybrid protein, elutes from DEAE-Sephadex in 0.37 M NaCl and is able to catalyze the hydrolysis of ATP to ADP + P(i) at a rate of 3 mumol/hr per mg. The remaining 10-15% of the D2 hybrid protein consists of form II which elutes from DEAE-Sephadex in 0.29 M NaCl and is able to hydrolyze ATP as well as to incorporate phosphorus from ATP into either the D2 hybrid protein itself or other protein acceptors such as phosvitin. Although both forms are able to bind DNA, the ATPase activity of form I cosediments with SV 40 DNA more efficiently than does the protein kinase activity of form II during glycerol gradient centrifugation. The ATPase activity of form I is efficiently inhibited by addition of anti-T gamma globulin to the reaction mixture whereas control gamma globulin has no effect. Similarly, the phosphorylation of the D2 hybrid protein by form II is inhibited by anti-T gamma globulin. By contrast, phosphorylation of phosvitin is specifically inhibited by antibody only when the immune complex is removed from the reaction mixture. Thus, it appears likely that one and possibly two enzymatic activities are carried out by the D2 hybrid protein. These findings are discussed in terms of mechanisms of SV 40 DNA replication and virally induced transformation.

Adenosine Triphosphatases↗
PubMed1978

The binding site on SV40 DNA for a T antigen-related protein.

A protein closely related to SV40 T antigen was purified in a biologically active form from cells infected with the defective adenovirus-SV40 hybrid, Ad2+D2. This 107,000 dalton hybrid protein binds and protects a specific portion of SV40 DNA from digestion by pancreatic DNAase I. Hybridization, endonuclease cleavage and pyrimidine tract analysis of the protected fragments reveal that the D2 hybrid protein binds in a sequential manner to tandem recognition sites which lie within a sequence of 120 nucleotides at position 67 near the origin of SV40 replication.

Adenoviridae↗
PubMed1978

Biological activity of purified simian virus 40 T antigen proteins.

Proteins related to simian virus 40 (SV40) T antigen uere isolated from cells infected with adenovirus 2/SV40 hybrids Ad2+D2 and Ad2+ND1 dp2 as well as from a line of human cells (SV80) transformed by SV40. The 96,000- and 107,000-dalton proteins of SV80 and Ad2+D2, after injection into the cytoplasm of cultured cells, rapidly accumulate in the nuclei, where they remain antigenically reactive for at least 20 hr and trigger DNA synthesis in quiescent cells. By contrast, the 23,000-dalton protein coded by Ad2+ND1 dp2 does not stimulate cellular DNA synthesis. However, all three purified proteins are able to provide helper function for the growth of adenovirus 2 in monkey cells. Thus, purified SV40 T antigen and proteins that share sequences with it retain the ability to carry out at least two functions associated with the product of the A gene of SV40.

Adenoviruses, Human↗
PubMed1977

Purification and comparative properties of the delta and sigma subunits of RNA polymerase from Bacillus subtilis.

Bacillus subtilis delta protein is a 21 500-Mr polypeptide that can be isolated in association with RNA polymerase holoenzyme from uninfected bacteria and with modified forms of RNA polymerase from cells infected with phage SP01 [Pero, J., Nelson, J. and Fox, T. (1975) Proc. Natl Acad. Sci. U.S.A. 72,1589]. Although no function has been assigned to delta protein in uninfected cells, this host polypeptide enhances the specificity of transcription by phage-modified forms of RNA polymerase that contain SP01-coded regulatory subunits. This report describes the purification of delta and sigma proteins from uninfected B. subtilis and examines the comparative effects of these polypeptides on transcription by core RNA polymerase. Purified sigma polypeptide was found to stimulate the transcription of phage DNA while having little effect on RNA synthesis with the synthetic DNA poly(dA-dT) as template. In contrast, purified delta protein markedly depressed the transcription of poly(dA-dT) while having little effect on enzyme activity with phage DNA as template. The inhibitory effect of delta protein on poly (dA-dT) transcription was strongly dependent on the presence of KC1 in the RNA synthesis reaction mixture.

Bacillus subtilis↗